Microbiological method of the biosynthesis of natural blue-violet colorants violacein and deoxyviolacein and the utilization thereof

ABSTRACT

Known microbiological methods are based upon the application of pigment-forming bacteria, in particular  Chromobacterium violaceum . The yield of these methods is very low, however, so that large industrial applications are not possible. However, in many fields of industry, there exists a very great demand for natural blue dyes which are substantially suitable for human digestion, which cannot be satisfied at present. Therefore, the method in accordance with the invention is characterized by the application of the newly discovered marine sediment bacterium  Pseudoalteromonas  species of the “Black Beauty” strain (originally deposited under file number DSM 13623 with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH., Braunschweig, Germany pursuant to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure). Compared to conventional methods, this marine sediment bacterium leads to an about thirteen-fold yield. An application of the natural dyes produced economically and by simple technical processes is thus possible in consumer and environmentally friendly products, in particular those from the food, textile or toy industries.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to a microbiological method for the biosynthesisof the natural blue-violet dyes violacein and deoxyviolacein from apigment-forming bacterium by cultivation of the bacterium, removal ofits cell mass by centrifuging, extraction of a raw dye extract from thecell mass and isolation of the dyes from the raw dye extract, and to theutilization of the dye produced by the microbiological method.

Dyes are of interest in more than one respect to microbiology and tochemistry. Since the ability to produce dyes is genetically fixed, theformation of dyes is also a first indication of the coincidentalformation of antibiotic agents. Microbial pigments are of greatstructural diversity. They may be derivatives of the material classes ofcarotenoids, phenazine dyes, pyrrole dyes, azaquinones, etc.

2. The Prior Art

For about the past twenty years, there has been an ongoing search in theUnited States of America and in Japan, with thermophilic bacteria fromdeep sea vents and symbiontic bacteria from invertebrate andvertebrates, for biotechnologically useful bioactive natural substances.In countries of the European Community the usefulness of marineorganisms has been recognized for the pharmaceutical industry, aquaculture, microbiological cleansing of oil contaminated areas of theocean as well as for development processes of biological films andbiological adhesion. However, in Germany it was not until three yearsago that projects were conceived on a larger scale, which involve marineorganisms in a more application-oriented direction of research. A largenumber of dyes are used in the textile, food, cosmetics andpharmaceutical industries. During recent years there has been apronounced trend of consumers preferring products which contain naturalsubstances. At present, yellow and red dyes are already being producedon an industrial scale from vegetable raw materials. However, in thementioned industrial fields there exists a large demand for natural blueand violet dyes. The search for a dye from this spectrum has taken on aparticular significance as a consequence of the legal prohibitionagainst the use in foods of the red-blue dye monascine, isolated fromMonascus purpureus. Classifying monascine as a food dye or colorant isnot possible because of allergological problems. A microbiologicallyproduced dye may thus help to close the gap and to open economicallyhighly interesting paths.

Pigments from the blue-violet spectrum are formed, among others, bymicroorganisms. The azaquinone indigoidine is formed by severalorganisms (Pseudonomas indigofera, Corynebacterium insidiosum,Arthrobacter arthrocyaneus and Arthrobacter polychromogenes) and issecreted into the surrounding medium. This is of advantage in respect ofa continuous process operation and of simplified product processing. Iteliminates the step of cell pulping. Indigoidine has been officiallyapproved as a food dye (E 132). Further approved food dyes from the bluedye spectrum are patent blue V (E 131), brilliant blue FCF and brilliantblack (E 151). The absorption spectra of these compounds 11e in therange of 570 nm to 638 nm and are, therefore, particularly suitable forapplications in food technology processes.

The blue-black pigment violacein, which is an indole derivative, wasfirst described in literature in 1882. It was then that a violet dye wasisolated from a bacterium which is now known as Chromobacteriumviolaceum. Heretofore, violacein could be isolated from the bacterialstrains of Janthinobacterium lividum, Chromobacterium lividum andAlteromanus luteoviolacea (see H. Laatsch et al.: “SpectroscopicProperties of Violacein and Related Compounds: Crystal Structure ofTetramethylviolacein”; J. Chem. Soc. Perkin Trans 2., 1984, pp.1331-1339). The structure of violacein was proved by synthesis in 1960.Chemically, violacein is characterized as3-[1,2-dihydro-5-(5-hydroxy-1H-indole-3-yl)-2-oxo-3-H-pyrrole-3-ylidene]-1,3-dihydro-2H-indole-2-on,of the summation formula C₂₀—H₁₃—N₃—O₃ (molecular weight 343.33). Themaximum absorption band, in a solution of methanol, is about 570 nm. Inwater, violacein is practically insoluble; but it is soluble in acetone,ethanol and dioxane. Violacein is the major component of a blue-violetpigment in Chromobacterium violaceum which is a microorganism taken fromthe soil and water of tropical regions, with deoxyviolacein occurring asa secondary component of identical structure, except that it has oneless oxygen atom (summation formula C₂₀—H₁₃—N₃—O₂). So-called “nativeviolacein” consists of a mixture of violacein with up to 10%deoxyviolacein. The violacein serves to protect the cell from radiationand to regulate the concentration of tryptophane below the toxic level.While violacein has antibiotic, antiviral and antitumoral properties, itdisplays no cyto-toxic or pathogenic effects. In more recentexaminations, violacein has also been used for dying textiles. Gooddying results have not only been obtained in connection with naturalfibers such as silk, wool and cotton, but also with synthetic fiberssuch as polyamide (see Shirata et al.: “Isolation of Bacteria ProducingBluish-Purple Pigment and Use for Dying”, Jpn. Agr. Res. Q 2000, 34(2),pp. 131-140, using pigment produced from Janthinobacterium lividum).

The biosynthesis from known microorganisms has been described in variouspublications. A method of producing native violacein, proceeding fromthe pigment-forming bacterium Chromobacterium violaceum, for use in thetreatment of viral diseases, is described in German patent specificationDE 3,935,066. The biosynthesis is based on the method steps of“cultivating the bacterium”, “removing the cell mass by centrifuging”,“extracting a raw dye extract from the cell mass”, and “isolating thedye from the raw dye extract”. The bacteria used as the starter materialare cultivated on a solid or liquid nutrient. Preferably, the bacteriaare cultivated in liquid nutrients since they can then be easilyseparated from their nutrient by centrifuging. The cultivation of thebacteria may be carried out with different process parameters infermentation tanks. In the known method, the grown bacterial bed, afterincubation, is separated and freeze-dried. Extraction of the rawviolacein is carried out in methanol in a Soxhlet extractor. Themethanol is removed by vacuum distillation. For isolating the violacein,the raw violacein is twice extracted by n-heptane, and is then filteredout. The residue is dissolved in a mixture of chloroform, acetone,pyridine 50:40:10. The mixture of the dye is then separated and purifiedby silica gel thin layer chromatography.

A simple method of obtaining highly purified violacein is known from thepaper “Production, Extraction and Purification of Violacein: AnAntibiotic Pigment Produced by Chromobacterium violaceum (Rettori,Duran, World Journal of Microbiology & Biotechnology 14 (1998), pp.685-688. To cultivate the bacterium, non-sterile cotton rags areinoculated with a suspension culture of the pigment-formingChromobacterium violaceum CCT 3496 and are stored in a stronglyventilated incubator where strong growth of the bacterium occurs.Thereafter, the cell mass is washed out of the cotton rags with asolvent, and is filtered to yield an extract of raw dye. A highlypurified violacein is isolated from this as a dye by different methods(Soxhlet extractor, high-performance liquid chromatography). However,the yield is relatively low and lies in the range of milligrams perliter of nutrient.

The production of violacein from L-tryptophan as a biosynthetic startermaterial is known from the paper “Biosynthesis of Violacein: A NovelRearrangement in Tryptophan Metabolism with 1,2-shift of the IndoleRing” (T. Hoshino et al., Agric. Biol. Chem. 51 (3), 1987, pp. 965-968,wherein the carbon skeleton of the pyrroleinone ring is formed bycondensation of the side groups of two tryptophan molecules by a1,2-shift of the indole ring. By comparison with the production ofviolacein from a pure suspension culture of Chromobacterium violaceumJCM 1249, it is possible to obtain an about 1.5-fold yield by addingL-tryptophan to the suspension culture. However, at the usual yield inthe range of milligrams per liter of nutrient, this is still to beconsidered as a very low yield.

In summary, the known methods of biosynthesis of violacein anddeoxyviolacein are not suitable for providing these dyes in greaterquantities. For use in the pharmaceutical field this is not absolutelynecessary, since in this context even the smallest quantities may beeffectively applied. In other applications, however, the industrialdemand for blue-violet dyes may be very large. If they are synthesizedby the known methods, the use of large quantities of suspension culturesis necessary, the processing of which will be correspondingly demandingas regards equipment and time.

OBJECT OF THE INVENTION

It is thus an object of the present invention to provide a method of thekind described supra by which significantly larger yields of violaceinand deoxyviolacein may be obtained economically and with simpleprocess-related technological equipment than by known methods. This, inturn, is to make possible particular applications with larger dyequantities.

BRIEF SUMMARY OF THE INVENTION

In the accomplishment of this object, the microbiological method of thebiosynthesis of the natural blue-violet violacein and deoxyviolaceindyes from a pigment-forming bacterium therefore provides for using thenewly discovered marine sediment bacterium Pseudoalteromonas species ofthe “Black Beauty” strain, which was first deposited, in accordance withthe Budapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure, under file numberDSM 13623 at the Deutsche Sammlung von Mikroorganismen und ZellkulturenGmbH., at Mascheroder Weg 1b, D-38124 Braunschweig, Germany (GermanCollection of Microorganisms and Cell Cultures GmbH. at Brunswick,Germany), which entails the occurrence of a yellow dye as aninsignificant secondary product, which is separated by a furtherextraction of the raw dye extract by dichloromethane.

Advantageous improvements of the inventive method in respect of theindividual method steps “cultivation of the bacterium”, “removal of thecell mass by centrifuging”, “extraction of the raw dye extract from thecell mass” and “isolating the dyes from the raw dye extract” may begleaned from the sub-claims and from the ensuing specification. Theblue-violet dyes violacein and deoxyviolacein whose biosynthesis inaccordance with the invention is economical and is carried out by simpletechnological processes may be applied particularly advantageously toproducts of the food, textile, pharmaceutical or toy industries whichfriendly to consumers as well as to the environment.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

A viable bacterium culture has been deposited at the InternationalDepository DSMZ—Deutsche Sammlung von Mikroorganismen und ZellkulturenGmbH., at MascheroderWeg 1b, D-38124 Braunschweig, Germany, inaccordance with the Budapest Treaty on the International Recognition ofthe Deposit of Microorganisms for the Purposes of Patent Procedure. On28 Jul. 2000 the applicant received from DSMZ a filing receipt oforiginal deposit, issued in accordance with Rule 7.1, and a Certificateof Viability issued in accordance with Rule 10.2. Thus, under filingnumber DSM 13623, the applicant obtained protection for the newlydiscovered marine sediment bacterium Pseudoalteromonas species of the“Black Beauty” strain, and for its applications. With the presentinventive method protection is claimed for the production off twoblue-violet dyes using this sediment bacterium and for its use inindustry (pigment and food industries) having a high demand for naturalblue dyes. As far as is known to applicant, it is the first time that amarine microorganism has been used for such purposes. Hitherto, dyesfrom marine bacteria have seldom been examined, so that this constitutea larger potential for the chemistry of natural substances.

The possible yield of violacein from the marine sediment bacteriumPseudoalteromonas species of the “Black Beauty” strain is in the rangeof thirteen times higher than with Chromobacterium violaceum. Forinstance, from 38.97 g of moist mass of “Black Beauty” bacteria oneobtains, after multiple extractions with hot methanol, a strongly violetcolored solution from which, after removal of the solvent, 2.1 g of ajet black raw extract may be isolated. Compared to known methods, theyield factor by the inventive method which is based upon thebiosynthesis of blue dyes from “Black Beauty”, is very high. The marinesediment bacterium “Black Beauty” thus contains a very high proportionof pigment which renders it particularly suitable for the production ofthe native dyes violacein and deoxyviolacein. Moreover, the yield ofpigment may yet be improved by optimizing the parameters at the variousmethod steps (dwell time, solvent ratios, etc.), in particular duringcultivation of the bacteria (nutrients, shaking frequency, oxygencharge, pH value, salt content, temperature, etc.). Overall theinventive method, at moderate process control and relatively lowinvestment in time, results in great economic advantages, especially inrespect of large scale technical uses. In this connection, it isparticularly meaningful in view of the human-friendly characteristics ofviolacein to apply the biotechnically produced dyes in fields ofindustry having a large demand for environmentally friendly blue dyes,in particular in the food, toy and textile industries.

Thin-layer chromatographic tests show that the raw dye extract from themarine sediment bacterium “Black Beauty” is composed of three pigmentfractions: a “weak” non-polar yellow dye, a further “weak” violet dye oflow polarity, as well as a “very strong” polar dye of a deep violetcolor. The deep violet dye amounts to about 90% of the raw dye extract.An analytic examination of the structure by high-resolution massspectrometry and multi-dimensional nuclear magnetic resonancespectroscopy has revealed that the deep violet dye is violacein and thatthe violet dye is deoxyviolacein. The main component of the yellow dyewhich appears in the raw dye extract as an insignificant secondaryproduct with a share of 2%, is palmitoleic acid. This a fatty acid whichis present as a subordinate secondary component in vegetable as well asanimal fats and oils. However, the yellow dye is not the subject of thepresent invention and requires further examinations regarding itsstructure. In the context of the invention, it is significant that itsproportion in the raw dye extract be very low, so that it does notnoticeably lower the yield of blue-violet dyes, and that it be easilyremovable from the raw dye extract to facilitate the productiontherefrom of the two blue-violet dyes.

The individual steps of the method in accordance with the invention willhereafter be set forth with greater particularity in connection withexemplary embodiments. However, other variations of parameters as knownfrom similar methods (see the cited state of the art, e.g. DE3,8813,465) are possible as well since the individual method steps areof a general nature and are known to persons of average skill in theart, which is, in fact, the basis for the simplicity of the method.

Cultivation of the Bacteria

The starter culture for the synthesis of the bio mass was permanentbacteria culture found at the Borkum Watt and stored since 1985 at −80°C. at the Alfred-Wegener-Institut fuer Polar-und Meeresforschung,Bremerhaven, Germany. It was thawed and cultivated in a marine culturebroth in four 2 liter Erlenmeyer flasks each container 1 liter ofnutrient solution at 25° C. on a rotary shaker (100 to 110 rpm). Afterfour days, the cells were harvested by centrifuging and the obtainedcell mass was used for the extraction of dye.

Isolation of the Raw Extract

38.97 g of the bacterial cell mass (wet) is placed in an Erlenmeyerflask and converted to a slurry with about 250 ml of hot methanol. Themixture is left undisturbed for about 2 hours, until the cell haveclearly precipitated. The supernatant is decanted, and the process isrepeated several times, until the cells remain as a colorless grey mass.This mass may be used, for instance, as an additive to animal feed. Thecombined methanolic extracts is separated from the solvent in a rotaryevaporator, and after drying in an oil pump vacuum, 2.10 g of a jetblack raw extract is obtained.

Isolation of the Yellow Dye

514 mg of the raw extract are suspended in 50 ml of dichloromethane andstirred in the dark in an argon atmosphere for about one hour. Theremaining solid residue (dye mixture) is separated, washed withdichloromethane and dried in an oil pump vacuum. After removal of thesolvent 29.8 mg of a yellow-brown oily liquid is isolated from themother liquor at the rotary evaporator. The liquid is columnchromatographed over silica gel with dichloromethane/acetate 1+1. Oneobtains 10.2 mg of a yellow liquid which crystallizes when cold.

Isolation of the Two Blue-Violet Dyes

73.5 mg of the dye mixture separated from the yellow dye are suspendedin 15 ml of dichloromethane/methanol (6+1) and left standing overnight.On the following morning the supernatant (13.5 mg) is chromatographedover 100 g silica gel with dichloromethane/methanol (6+1). One obtains0.4 mg deoxyviolacein and 2.9 mg violacein. For analytical purposes, thelatter may be additionally purified by preparative HPLC as well as byisothermal fractional crystallization from methanol/n-hexane.

The afore-mentioned steps of preparation and examination are part of themaster's thesis “Isolation and Explanation of the Structure of Pigmentsfrom Marine Sediment Bacteria” (Diplomarbeit “Isolierung undStrukturaufklaerung von Pigmenten aus marinen Sedimentbakterien”) whichwas presented to the Institute Administration for Organic Chemistry,Resort 2, of the University of Bremen, Germany. If required, furtherdetails regarding individual process and examination parameters andresults may be taken from the thesis after its publication at thebeginning of 2001. There also exists an application for support of theproject during the time between 1 Apr. 2001 and 31 Mar. 2002 by the BMBF(German Federal Ministry for Education and Research), Biology, Energy,Environment (BEO) at the research center at Juelich, resort for oceanand polar research, geo sciences, relating to the subject “Research ofMarine Native Substances—Application of native dyes from marine sedimentbacteria”. In this connection, participation by industrial partners isinvited.

1. A microbiological method of the biosynthesis and isolation of naturalblue-violet dyes violacein and oxyviolacein, comprising the steps of:providing a predetermined quantity of the marine sediment bacteriumPseudoalteromonas species of the “Black Beauty” strain DSM 13623, whichproduces said blue-violet dyes and a yellow dye occurring therein as ana secondary product; cultivating the bacterium on a nutrient broth toobtain a culture; centrifuging the culture to obtain a cell mass;extracting a raw dye extract comprising said blue-violet dyes and saidyellow dye from the cell mass; and isolating said blue-violet dyes fromthe raw dye extract.
 2. The method of claim 1, further comprising thestep of extracting the yellow dye from the raw dye extract withdichloromethane.
 3. The method of claim 1, wherein the step ofextracting the raw dye extract from the cell mass further comprises thesteps of: forming a slurry of the cell mass in methanol; precipitatingthe cell mass from said methanolic slurry for about 2 hours andseparating the resultant supernatant which comprises a methanolicextract of the dye; repeating the steps of forming a slurry to separatesaid supernatant from the precipitated cell mass until the cell massremains as a gray mass; removing solvents by subjecting the supernatantto a rotary evaporator to obtain a concentrated methanolic extract; anddrying said concentrated methanolic extract in an oil pump vacuum. 4.The method of claim 2, wherein the step of removing the yellow dye iscarried out in the dark in an argon atmosphere by stirring a suspensioncomprising the raw dye extract and dichloromethane causing the yellowfraction to dissolve and the blue-violet dyes to occur as a mixture ofsolids.
 5. The method of claim 1, wherein the step of isolating the dyesfrom the raw dye extract further comprises the steps of: suspending theraw dye extract in a solvent mixture of 6 parts dichloromethane and 1part methanol for about 12 hours to obtain a supernatant and aprecipitate; decanting the supernatant; and subjecting the supernatantto column chromatography over silica gel with a mixture of 6 partsdichloromethane and 1 part methanol to obtain violacein.
 6. The methodof claim 5, further comprising the step of: subjecting said isolatedviolacein to high pressure chromatography to obtain purified violacein.7. The method of claim 5, further comprising the step of: purifying theisolated violacein by isothermal recrystallization from a mixture ofmethanol and n-heptane.